Effect of androgens on histochemical fibre type

Summary The temporal muscles of the guinea pig show a sexual differentiation reflected in their histochemical enzyme pattern. Using histochemical methods for mitochondrial (SDH, -GPDH), and glycolytic enzymes (phosphorylase, LDH) it could be shown, that in adult animals the male muscle is a white muscle with marked activity of glycolytic enzymes, the female muscle a red muscle displaying high activity of mitochondrial enzymes. This differential enzyme pattern can be converted by the application of testosterone to the female type during the postnatal development. The male sex hormone thus affects the histochemical enzyme pattern of the muscle, converting the red, female into a white, male muscle in the female guinea pig.     

Multiple Phosphorylation Sites and Quaternary Organization of Guanine-Nucleotide Exchange Complex of Elongation Factor-1 (EF-1βγδ/ValRS) Control the Various Functions of EF-1α

The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1 (EF-1), comprises four different subunits including valyl-tRNA synthetase (EF-1/ValRS). The factor is multiply-phosphorylated by three different protein kinases, protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDK1). EF-1/ValRS is organized as a macromolecular complex for which we propose a new structural model. Evidence that EF-1/ValRS is a sophisticated supramolecular complex containing many phosphorylation sites, makes it a potential regulator of any of the functions of its partner EF-1, not only involved in protein synthesis elongation, but also in many other cellular functions.     

Rapid detection of single nucleotide deletions: application to the β 6 (-A) mutation of the β-globin gene and to cystic fibrosis

The formation of heteroduplexes from the amplified products of homologous alleles has been shown to be useful in the identification of heterozygotes carrying deletion or insertion mutations. Here, we describe an improved procedure that allows the detection of single base pair (bp) deletions on nondenaturing polyacrylamide gels. Carriers for a common Mediterranean -thalassemic mutation, 6 (-A), could be easily detected by use of this method, as could carriers of a 1-bp deletion in the cystic fibrosis gene.     

Filipino β-thalassemia due to a large deletion: identification of the deletion end points and polymerase chain reaction (PCR)-based diagnosis

The gene frequency of -thalassemia among Filipinos is estimated to be 0.02, although little is known about the mutations involved. Recently, an extensive -thalassemia deletion was reported in several unrelated individuals of Filipino descent. The deletion begins approximately 4 kb upstream of the -globin gene, and extends 3 beyond the -globin gene. In this report, we identify the 5 and 3 deletion endpoints and present a polymerase chain reaction (PCR) strategy for rapid DNA diagnosis of the Filipino -thalassemia deletion.     

Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules

Summary The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) Viable cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of 41. Their measured Ca content is 4.1±1.4 (sem) mmol/kg dry wt in the cytoplasm and 3.1 ± 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of 3.8 mmol/kg dry wt. 2) Nonviable cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of 1. The Ca content is 15±2 mmol/kg dry wt in the cytoplasm and 685±139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions ( 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in toal Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusion in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that thein situ mitochondrial Ca content of viable cells from the kidney, proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

Over expression of integrin α 5 β 1 in human hepatocellular carcinoma cell line suppresses cell proliferation in vitro and tumorigenicity in nude mice

Integrin 5 1 and 2 1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin 5 1 was decreased and another integrin 6 1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singlely transfected with integrin 5 and/or 1 cDNAs were established, and designated 5 1.6-7721, 5.3-7721, and 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin 5 and 1 subunits resulted in the overexpression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in 5.3-7721, 1.6-7721, 5 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with 1.6-7721,and 5 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin 5 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin 5 1 in malignant cells could be a potential means of treating hepatocellular carcinoma.     

Distribution of carbon isotope composition of modern soils on the Qinghai-Tibetan Plateau

This paper presents a large data set on carbon isotope composition (¹³C) of modern soils which were collected under the main vegetation communities along an altitude of 1250–5500m above sea level in the Qinghai-Tibetan Plateau. The ¹³C values of 198 samples range from –28.6 to –15.1 versus PDB and exhibit a clean relation to different vegetation communities from forest (–25.9±1.2) to shrub (–24.7±1.4), steppe (–23.1±1.3), alpine meadow (–23.6±0.7), alpine desert steppe (–21.3±1.6), and alpine desert (–18.9±2.5). We attributed the observed variability in ¹³C values to that the mean annual precipitation (MAP) and the mean annual temperature (MAT) are the main factors controlling the distribution of vegetation types in the Tibetan Plateau, which causes the change in carbon isotope composition of modern soils at any given altitude. The result of both linear and nonlinear regression analyses also confirms that MAP and MAT are the major factors affecting the ¹³C values of surface soils. In the absence of favorable moisture and temperature conditions, low pCO₂ alone is not sufficient to cause the distinct changes in carbon isotope composition of modern soils in the Tibetan Plateau. This study provides some fundamental information on the carbon isotope composition of terrestrial carbon pools and bears some practical significance for the use of carbon isotope data to document vegetation changes and environmental conditions of the high plateau in the past.     

An isolated β 1 exon next to the DR α gene in the HLA-D region

A cosmid clone containing the DR gene and a ₁ exon of a DR -related gene was isolated from a human cosmid clone bank made from the consanguineous DR7 cell line MANN. No other DR-related exons were found on this clone. The ₁ exon was located about 15 kb away from the DR gene in a tail-to-tail (3 to 3) orientation. The exon contained several deleterious mutations: a defective splice site at the 5 end, two translational frame shifts (a 1 by deletion and a 1 bp insertion), and three extra cysteine residues. Nucleotide and amino acid sequence comparisons of the ₁ exon indicated that although it is substantially different from other class II -chain genes, it is slightly more related to DR than to any other class 11 gene. The DR-related sequence was on a DNA fragment which showed no polymorphism on a panel of cell lines with Eco RI or Pst 1. These Southern blots, however, revealed a related, polymorphic sequence in the human genome. Nucleotide sequences in the intron flanking the ₁ exon shared greater sequence homology than the ₁ exon itself when compared with the DR genomic sequence. The exon may play a role in the generation of variation in expressed class II -chain genes and it may be a relic of a different subset of class II products.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

β-Thalassemia mutations in Singapore — a strategy for prenatal diagnosis

The strategy for early prenatal diagnosis of -thalassemia in Singapore by direct detection of the mutant -globin gene requires the spectrum of mutations producing the disorder in this population to be characterized. We analyzed 134 -thalassemia alleles from Singapore by specific oligonucleotide hybridization after DNA amplification, using a nonradioactive enhanced chemiluminescence detection system. The mutations were identified in 90% of the alleles using five oligonucleotide probes for the following mutations: codons 41/42 (deletion -TCTT), IVS II nt 654 (CT), codon 17 (AT), IVS I nt 5 (GC), and -28 TATA box (AG). Together with the strategy of direct sequencing, a total of 97% of the mutations were identified. In the Chinese subpopulation, 97% of the mutations were detected by the oligonucleotide probes. Using just four oligonucleotide probes would identify 96% of the mutations, and 76% of the mutations were accounted for by codon 41/42 (-TCTT) and IVS II nt 654 (CT) mutations. Thus in this subpopulation early prenatal diagnosis would be possible in virtually all the affected families.     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

Seasonal succession,standing crop and determinants of primary productivity of the phytoplankton of Ministik Lake,Alberta, Canada

Ministik Lake (longitude 113°01; latitude 53°21), a well-mixed, shallow (mean depth 1.83 m), eutrophic lake is characterized by eutrophic chlorococcalean and cyanophycean phytoplankton associations, and little change in standing crop size with increasing depth. Standing crops and primary productivity are low during the winter but pronounced spring and late summer/early autumn maxima occur. Mean yearly areal standing crop (B) and primary productivity (A) were 199.2 mg m^–2 chlorophyll a and 319.5 mg C hr^–1 m^–2 respectively. Annual productivity was estimated at 1399,6 g C m^–2yr^–1. The mean increase in the extinction coefficient () per unit increase in standing crop (B) was 0.03 In units m^–1. High non-algal light attenuation (q) occurred averaging 46% which prevented the ratio B from attaining more than 74.2% of the theoretical maximum except once when selfshading occurred. Insignificant relationships existed between B (mg m^–3 chlorophyll a) and A_max (mg C hr^–1 m^–3), A and B and A and B. Close correlations existed between A and A_max/ and A and I₀ (W m^–2). The depth of the euphotic zone (Z_eu) varied between 0.6 and 2.0 m; the average relationship between z_eu and was z_eu = 3.78/_te, and the mean standing crop in the euphotic zone represented 58.3% of the theoritical maximum. The high _q values made the model of Talling (1957) inapplicable to this lake. The Q₁₀ value for the lake was 1.20. _max (mg C chlorophyll a ^–1 hr^–1) was closely related to both temperature and irradiance, and depressed by high pH values.Growth of the cyanophycean algae was correlated with temperature, chlorophycean algae with phosphate-phosphorus and temperature and the diatoms with dissolved silica and phosphate-phosphorus, but only in the case of Chaetoceros elmorii did any nutrient appear limiting. Indirect evidence that free CO₂ limited photosynthetic rates is provided by the _max:pH relationship.     

The minimal polymorphism of class II Eα chains is not due to the functional neutrality of mutations

Given the extensive allelic amino acid sequence polymorphism present in the first domain of A, A, and E chains and its profound effects on class II function, the minimal polymorphism in the mouse E chain (and in its human homologue DR) is paradox. Two possible explanations for the lack of polymorphism in E are: (1) the E chain plays such a uniquely critical structural/functional role in antigen presentation, T-cell activation, repertoire selection, and/or pairing with E or other proteins for expression that it cannot vary, and mutations are selected against; (2) the E chain plays a less significant role than the outer domains of other major histocompatibility complex (MHC) proteins in determining the interactions with processed peptides or with T-cell receptor (TCR), so there is no selective pressure to maintain new mutations. To explore this question we compared the ability of transfectants expressing wild type (wt) EE ^d and mutant E wt E ^d proteins to present peptides and bacterial superantigens to T-cell hybridomas. Mutations at the E amino acid positions 31, 52, and 65&66, to residues that represent allelic alternatives in A chains, significantly reduced activation of peptide-specific T hybridomas, and mutations at 71 sometimes enhanced T-cell stimulation. None of the E mutations reduced, and some enhanced, superantigen stimulation of T-cell hybridomas. These results argue against the hypothesis that E chains are minimally polymorphic because mutations in E are functionally neutral.     

Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8, C8, C8) encoded by separate genes. C8 and C8 are linked on chromosome 1p32, whereas C8 is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 gene. The gene contains at least 11 exons spanning 70kb of DNA. When compared to C6, C8 and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 and C8 linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5kb) and in a 5- 3 orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.     

Developmental changes in neutral glycosphingolipids of mouse placenta

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive spot migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of¹²⁵I labelledHelix pomatia agglutinin (-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.Abbreviations asialo-GM₁ Gal 3GalNAc4Gal4Glc1Cer - BCIP 5-bromo-4-chloro-3-indolylphosphate - DHC lactosylceramide, Gal4Glc1Cer - Forssman antigen GalNAc3GalNAc3Gal4Gal4Glc1Cer - globoside GalNAc3Gal4Gal4Glc1Cer - GSL glycosphingolipids - HPA Helix pomatia agglutinin - HPTLC high performance thin layer chromatography - MHC galactosylceramide, Gal1Cer - MHC glucosylceramide, Glc1Cer - PBS phosphate-buffered saline - PNA peanut agglutinin - PVP poly(vinylpyrrolidone), mol. wt 40 000 - SBA soybean agglutinin - THC trihexosylceramide, Gal4Gal4Glc1Cer. To whom correspondence should be addressed.     

Novel promoter and splice junction defects add to the genetic,clinical or geographic heterogeneity of β-thalassaemia in the Portuguese population

Summary In order to delineate the spectrum and the relative abundance of -globin gene defects causing thalassaemia in the Portuguese population, a representative sample was analysed including 51 -thalassaemia carriers along with 26 patients representing different clinical phenotypes. Seven mutations were identified, four of which [codon 39 (CT), 39%; intervening sequence (IVS)1 nucleotide (nt) 1 (GA), 26%; IVS1 nt 110 (GA), 17%; IVS1 nt6 (TC), 15%] account for 97% of 93 -thalassaemia chromosomes. Two previously undescribed mutations, namely a CT substitution at position — 90 in the proximal CACCC box, and the deletion of nucleotides 4 and 5 (AG) in IVS 2 were identified. The uncommon, though ubiquitous, GT transversion at codon 121 was found once upon haplotype V. Direct prenatal diagnosis can be offered to 95% of couples at risk of bearing a thalassaemic child.